Structural study of a Kv channel in different conformations in membranes Voltage-gated ion channels are important membrane proteins that play crucial roles in many cellular events. Understanding how these channels operate in membranes is important not only for the biophysical elucidation of their function but also for the development of pharmacological treatment for human diseases related to the dysfunction of various voltage-gated ion channels. Despite extensive structural and functional studies in the past, there are still open questions on the molecular mechanism of the voltage-dependent gating. Recent discoveries demonstrated that voltage-gated potassium (Kv) channels have strong interactions with phospholipid membranes, and their functions are modulated by their lipid environments. But all available structures of voltage-gated ion channels are in detergents or mixed detergent/lipid micelles. We hypothesize that the strong protein-lipid interactions affect both the structure and function of voltage-gated ion channels, and that elucidating the structural details of the voltage-dependent gating requires structures of a voltage-gated ion channel in different conformations in lipid bilayers. In this proposal, we will use as a model system the KvAP, a Kv channel from Aeropyrum pernix, to examine this hypothesis by characterizing the channel functions in different lipids and obtaining its structures in different conformations. The KvAP is a good model system because the protein is much more stable than recombinant eukaryotic channels, and can be readily reconstituted into membrane systems of different lipid composition. Our Aim 1 will focus on characterizing the UP conformation of the KvAP voltage sensor in conditions similar to those used in our two- dimensional crystallization, and then obtaining the structure of the channel in such a conformation. Our Aim 2 will use biochemical and electrophysiological assays to characterize conditions that favor the KvAP voltage sensor in its DOWN conformation, and apply these conditions to screen for 2D crystals of the channel, which will pave the way towards structure determination. Results from our two-pronged studies will offer new evidence to address the fundamental questions on voltage-dependent gating. Because of the well-conserved structural features in the superfamily of voltage-gated ion channels, our results will have general implications for eukaryotic voltage-gated ion channels. PUBLIC HEALTH RELEVANCE: Structural study of a Kv channel in different conformations in membranes Voltage-gated ion channels play essential roles in many physiological activities. They have been shown to require phospholipid bilayers for their proper function. Their strong interactions with phospholipids suggest that structures of the channel proteins in membranes are necessary for elucidating the structural details on voltage-dependent gating. In this proposal, a voltage-gated potassium (Kv) channel will be studied in two explicitly different conformations in membranes. Structural results will provide novel insights on how the voltage-gated ion channels are structurally adapted to their native lipid environment and how they rearrange themselves to achieve voltage-dependent gating.